The Signal Transducer and Activator of Transcription 3 (STAT3) is a transcription factor that regulates many biological processes including cell proliferation, differentiation and survival (Debnath, et al., Small Molecule Inhibitors of Signal Transducer and Activator of Transcription 3 (Stat3) Protein. Journal of Medicinal Chemistry 2012, 55, 6645-6668; Masciocchi, et al., Signal transducer and activator of transcription 3 (STAT3): a promising target for anticancer therapy. Future Medicinal Chemistry 2011, 3, 567-597; Lavecchia, et al., Novel inhibitors of signal transducer and activator of transcription 3 signaling pathway: an update on the recent patent literature. Expert Opin Ther Pat 2014, 24, 383-400; Page, et al., Signal transducer and activator of transcription 3 inhibitors: a patent review. Expert Opin Ther Pat 2011, 21, 65-83). Under normal physiological conditions, the activation of STAT3 is transient and tightly regulated, and is only triggered by the stimulation of extracellular cytokines and growth factors such as IL-6, EGF and PDGF, which leads to the phosphorylation of a specific tyrosine (Y-705) on STAT3 (Sun, et al., Cucurbitacin Q: a selective STAT3 activation inhibitor with potent antitumor activity. Oncogene 2005, 24, 3236-3245; Turkson, et al., Novel peptidomimetic inhibitors of Stat3 signaling and oncogenesis. European Journal of Cancer 2002, 38, S98-S98). This phosphorylation subsequently induces the dimerization of STAT3-STAT3 which is stabilized by two reciprocal phosphotyrosine-SH2 binding interactions. The phosphorylated STAT3 dimers translocate to the cell nucleus and bind to promoter regions in DNA, resulting in regulation of specific gene expression (Zhang, et al., A Novel Inhibitor of STAT3 Homodimerization Selectively Suppresses STAT3 Activity and Malignant Transformation. Cancer Research 2013, 73, 1922-1933; Zhang, et al., Orally bioavailable small-molecule inhibitor of transcription factor Stat3 regresses human breast and lung cancer xenografts. Proceedings of the National Academy of Sciences of the United States of America 2012, 109, 9623-9628). However, STAT3 is constitutively activated in a variety of cancers including both solid tumors (i.e. breast, prostate, lung, pancreatic) and hematological cancers (i.e. lymphoma, leukemia, melanoma) (Urlam, et al., Development of new N-arylbenzamides as STAT3 dimerization inhibitors. Medchemcomm 2013, 4, 932-941; Siddiquee, et al., An Oxazole-Based Small-Molecule Stat3 Inhibitor Modulates Stat3 Stability and Processing and Induces Antitumor Cell Effects (vol 2, pg 787, 2007). Acs Chemical Biology 2009, 4, 309-309; Cheng, et al., Stat3 Inhibition Augments the Immunogenicity of B-cell Lymphoma Cells, Leading to Effective Antitumor Immunity. Cancer Research 2012, 72, 4440-4448). Such hyper-activation of STAT3 leads to uncontrolled cell proliferation by activating cell cycle regulators such as c-Myc and cyclin D1, and enhancement of cell survival by selectively inducing the expression of anti-apoptotic proteins including Bcl-xL and survivin. As such, STAT3 mediated signaling pathways are recognized as valid cancer targets.
Many approaches have been adopted to inhibit constitutive activation of STAT3. Among the domains of STAT3 that regulate its function are SH2 domain (dimerization domain) and the DNA-binding domain, seen in FIG. 1. Thus, STAT3 signaling can be suppressed by either inhibition of STAT3 dimerization or STAT3-DNA binding (Debnath, et al., Small Molecule Inhibitors of Signal Transducer and Activator of Transcription 3 (Stat3) Protein. Journal of Medicinal Chemistry 2012, 55, 6645-6668; Masciocchi, et al., Signal transducer and activator of transcription 3 (STAT3): a promising target for anticancer therapy. Future Medicinal Chemistry 2011, 3, 567-597; Lavecchia, et al., Novel inhibitors of signal transducer and activator of transcription 3 signaling pathway: an update on the recent patent literature. Expert Opin Ther Pat. 2014, 24, 383-400; Page, et al., Signal transducer and activator of transcription 3 inhibitors: a patent review. Expert Opin Ther Pat 2011, 21, 65-83). Significant effort has been devoted to the development of STAT3/STAT3 dimerization inhibitors that disrupt the phosphotyrosine-SH2 binding (Zhang, et al., A Novel Inhibitor of STAT3 Homodimerization Selectively Suppresses STAT3 Activity and Malignant Transformation. Cancer Research 2013, 73, 1922-1933; Zhang, et al., Orally bioavailable small-molecule inhibitor of transcription factor Stat3 regresses human breast and lung cancer xenografts. Proceedings of the National Academy of Sciences of the United States of America 2012, 109, 9623-9628; Fletcher, et al., Antagonism of the Stat3-Stat3 Protein Dimer with Salicylic Acid Based Small Molecules. Chemmedchem 2011, 6, 1459-1470; Siddiquee, et al., An oxazole-based small-molecule Stat3 inhibitor modulates Stat3 stability and processing and induces antitumor cell effects. Acs Chemical Biology 2007, 2, 787-798; Gunning, et al., Targeting Protein-Protein Interactions: Suppression of Stat3 Dimerization with Rationally Designed Small-Molecule, Nonpeptidic SH2 Domain Binders. Chembiochem 2008, 9, 2800-2803; Mandal, et al., Synthesis of phosphatase-stable, cell-permeable peptidomimetic prodrugs that target the SH2 domain of Stat3. Org Lett 2009, 11, 3394-7; Mandal, et al., Potent and Selective Phosphopeptide Mimetic Prodrugs Targeted to the Src Homology 2 (SH2) Domain of Signal Transducer and Activator of Transcription 3. Journal of Medicinal Chemistry 2011, 54, 3549-3563; Mandal, et al., Structure-Activity Studies of Phosphopeptidomimetic Prodrugs Targeting the Src Homology 2 (SH2) Domain of Signal Transducer and Activator of Transcription 3 (Stat3). International Journal of Peptide Research and Therapeutics 2013, 19, 3-12). Because STAT3-DNA binding is downstream of phosphorylated STAT3 dimerization, most STAT3 dimerization inhibitors also exhibit inhibitory activity against STAT3-DNA binding (Debnath, et al., Small Molecule Inhibitors of Signal Transducer and Activator of Transcription 3 (Stat3) Protein. Journal of Medicinal Chemistry 2012, 55, 6645-6668; Masciocchi, et al., Signal transducer and activator of transcription 3 (STAT3): a promising target for anticancer therapy. Future Medicinal Chemistry 2011, 3, 567-597; Lavecchia, et al., Novel inhibitors of signal transducer and activator of transcription 3 signaling pathway: an update on the recent patent literature. Expert Opin Ther Pat 2014, 24, 383-400; Page, et al., Signal transducer and activator of transcription 3 inhibitors: a patent review. Expert Opin Ther Pat 2011, 21, 65-83). However, molecules that specifically recognize the STAT3 DNA binding domain, and therefore directly disrupt STAT3-DNA binding interactions, are rare. This is because the STAT3-DNA binding interface is large and unlike in other transcription factor/DNA interactions the STAT3 DNA binding domain is complex involving residues from multiple α-helices and β-sheets (Levy, & Darnell, Stats: transcriptional control and biological impact. Nat Rev Mol Cell Biol 2002, 3, 651-62). As such, the rational design of inhibitors is difficult. However, since a recent discovery suggested that phosphorylation is not required for nuclear transport of STAT3 (Nkansah, et al., Observation of unphosphorylated STAT3 core protein binding to target dsDNA by PEMSA and X-ray crystallography. Febs Letters 2013, 587, 833-839) and subsequent DNA binding, disruption of STAT3-DNA binding may be an alternative approach in the regulation of gene transcription compared to the inhibition of SH2 domain dimerization. Buettner, et al. (Buettner, et al., Alkylation of cysteine 468 in Stat3 defines a novel site for therapeutic development. ACS Chem Biol 2011, 6, 432-43) used a virtual screening to identify NSC-368262 that inhibits STAT3-DNA binding by covalently alkylating Cys468, a residue on the DNA-binding surface of STAT3.
The exploration of new and non-covalent molecular ligands that selectively inhibit STAT3-DNA binding are therefore very significant, as such an effort will not only lead to novel anti-cancer therapeutics, but also provide a new tool to further dissect the functional role of STAT3 in the regulation of cell proliferation and apoptosis. However, the art is currently underdeveloped in this field. As such, novel STATS-DNA binding inhibitors are required for therapeutic and academic use.